The overall goal of this project in this Program is to understand the cellular and molecular mechanisms by which induction of islet and skin graft tolerance is produced through the combination of donor-specific transfusion and anti-CD154 therapy. T cell deletion mechanisms as well as the induction of antigen-specific anergy of CD8+ and/or CD4 + T cells are likely involved. Our approach is to first identify and characterize helper T lymphocyte proteins that confer and maintain the anergic state induced in vitro through TCR stimulation (signal 1), and secondly to identify which of these proteins may play a direct role in graft tolerance induction in vivo. The latter goal is pursued in collaboration with Program investigators in projects 1 and 2 using animal model systems to study the cell types and mechanisms involved in both islet and skin graft tolerance. To accomplish the first goal, we have employed two genomics-based methods-- suppression subtractive hybridization and Affymetrix GeneChip microarrays--to identify genes highly expressed in cultured A.E7 Thl CD4 + cells at different times after induction of anergy, compared to gene expression levels in unstimulated or fully co-stimulated A.E7 T cells. Sets of such genes based on RNA extracted from anergized A.E7 T cells at 12 hours and 2 or 5 days after anergy induction have been generated in our laboratory over the past year. This approach is more powerful than the directed study of known proteins in that it reduces bias from predetermined hypotheses. Already several genes have been found to exhibit high expression levels specifically in anergic T cells, and appear to be excellent candidates for further study. These include PAC1, a dual specificity protein ser/thr phosphatase that negatively regulates ERK protein kinases known to be required for T cell activation. Another exciting new candidate gene we discovered is Egr2, an early response transcription factor known to regulate Fas ligand expression in T cells. After a brief initial increase following treatment of AE.7 cells with either anti-CD3 alone or anti-CD3 plus anti-CD28, Egr2 expression becomes highly restricted to the anergic state in AE.7 cells. We now have preliminary data indicating Egr2 is also elevated in CD4 + T cells obtained from lymph nodes draining skin allografts in mice tolerized with anti-CD154 plus donor cells. Also highly and selectively expressed in anergic T cells is GRP1, an Arf 6 exchange factor we originally cloned as a target of PI-3 kinase. We propose to analyze potential functions of these and other candidate proteins in conferring A.E7 T cell anergy by using methods that will specifically block or attenuate their expression, including antisense RNA, RNAi, and dominant inhibitory approaches. For testing the potential role of these candidate proteins in animal models of islet and skin graft tolerance, gene knockout mice will be generated. We have already succeeded in producing the first of these--the GRP1 gene-ablated mouse--which remains viable and will be used for such studies on islet and skin engraftment. We believe this overall approach represents a powerful strategy that complements other projects in this Program to elucidate the mechanisms underlying graft tolerance in intact animals.